# US 5,346,824 A · DNA encoding variants of tissue plasminogen activators and expression vectors and hosts thereof

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- Number: US 5,346,824 A
- Document ID: US5346824A
- Status: Granted
- Kind: A
- Application number: 08/036,014
- Filed: 1993-03-22
- Granted: 1994-09-13
- Earliest claimed: 1988-05-20
- Assignees: Genentech, Inc.
- Inventors: Stephen Anderson; Kevin M. Brady; Bruce A. Keyt; Leonard G. Presta
- Web page: https://patentgrep.com/patent/US5346824A

## CPC

- C12N 9/6459: {t-plasminogen activator (3.4.21.68), i.e. tPA}
- C12Y 304/21069: Protein C activated (3.4.21.69)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

A fibrinolytically active amino acid sequence variant of a plasminogen activator is prepared that has one or more glycosylation sites in regions that are not glycosylated in the native molecule. DNA sequences can be prepared that encode the variants, as well as expression vectors incorporating the DNA sequences, and host cells transformed with the expression vectors. The variants may be used in a pharmaceutical preparation to treat a vascular disease or condition in patients.

## Claims

### Claim 1

A DNA molecule encoding a fibrinolytically active human tissue plasminogen activator (t-PA) amino acid sequence variant having one or more amino acid substitutions which provide an Asn-X-Ser or Asn-X-Thr tripeptidyl sequence that starts at an amino acid position selected from the group consisting of amino acid positions 57 to 61, 63 to 69, 99, 101, 103 to 105, 106, 107, 109, 112 and 250 of the amino acid sequence of native human t-PA, wherein X is any amino acid except proline.

### Claim 2 (depends on 1)

A replicable expression vector containing the DNA molecule of claim 1 and capable, in a suitable transformant host cell, of expressing said DNA molecule.

### Claim 3 (depends on 2)

A recombinant host cell transformed with the vector of claim 2.

### Claim 4 (depends on 3)

The cell of claim 3 that is a eukaryotic cell.

### Claim 5 (depends on 4)

The cell of claim 4 that is a yeast cell.

### Claim 6 (depends on 4)

The cell of claim 4 that is a mammalian cell.

### Claim 7 (depends on 6)

The cell of claim 6 that is from a Chinese hamster ovary cell line.